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Abstract

Objective

Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect
in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety,
which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less
immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely
overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third
generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and
cytotoxicity of redirected human T cells.

Materials and Methods

In this experimental study, we established a second generation novel CAR (VHH-based anti-
MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane
domain and signalling endodomains of CD28 and CD3+. Next, we constructed lentiviral vectors that contained this
CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell
surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+T cells.

Results

The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR
showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic
activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared
with A431 (MUC1-negative) or untransduced T cells.